U.S. patent application number 10/495594 was filed with the patent office on 2005-10-06 for detection of predisposition to osteoporosis.
Invention is credited to Bansal, Aruna, Reed, Peter Wayne, Sallstrom, Eva Gunvor Kristina, Wilson, Scott Geoffrey.
Application Number | 20050221306 10/495594 |
Document ID | / |
Family ID | 9925099 |
Filed Date | 2005-10-06 |
United States Patent
Application |
20050221306 |
Kind Code |
A1 |
Wilson, Scott Geoffrey ; et
al. |
October 6, 2005 |
Detection of predisposition to osteoporosis
Abstract
The invention provides novel reagents, kits, and methods for
diagnosis of predisposition to low spine bone mineral density, low
total hip bone mineral density, low femoral neck bone mineral
density, or osteoporosis, based on analysis of polymorphic variants
of the nucleic acid set forth in SEQ ID NO:1.
Inventors: |
Wilson, Scott Geoffrey;
(Ocean Reef, AU) ; Sallstrom, Eva Gunvor Kristina;
(Uppsala, SE) ; Reed, Peter Wayne; (Rotorua,
NZ) ; Bansal, Aruna; (Hertfordshire, GB) |
Correspondence
Address: |
BIOTECHNOLOGY LAW GROUP
c/o PORTFOLIO IP
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Family ID: |
9925099 |
Appl. No.: |
10/495594 |
Filed: |
April 14, 2005 |
PCT Filed: |
October 24, 2002 |
PCT NO: |
PCT/GB02/04809 |
Current U.S.
Class: |
435/6.11 ;
435/287.2; 435/6.17; 536/24.3 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/6883 20130101; C12Q 2600/172 20130101 |
Class at
Publication: |
435/006 ;
435/287.2; 536/024.3 |
International
Class: |
C12Q 001/68; C12M
001/34; C07H 021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 3, 2001 |
GB |
0126436.5 |
Claims
1. A microarray comprising at least one oligonucleotide
complementary to a polymorphic region within a nucleic acid having
a sequence as set forth in SEQ ID NO:1, wherein the region
corresponds to a polymorphic site selected from the group
consisting of position 245 of SEQ ID NO:1 and position 1470 of SEQ
ID NO:1.
2. The microarray of claim 1, comprising an oligonucleotide
complementary to a polymorphic region corresponding to position 245
of SEQ ID NO:1 and an oligonucleotide complementary to a
polymorphic region corresponding to position 1470 of SEQ ID
NO:1.
3. An oligonucleotide complementary to a polymorphic region within
a nucleic acid having a sequence as set forth in SEQ ID NO:1,
wherein the region corresponds to a polymorphic site selected from
the group consisting of position 245 of SEQ ID NO:1 and position
1470 of SEQ ID NO:1.
4. The oligonucleotide of claim 3, wherein the region comprises a
sequence selected from the group consisting of SEQ ID NO:2; SEQ ID
NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID
NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:12; SEQ ID
NO:13; SEQ ID NO:14; SEQ ID NO:15; SEQ ID NO:16; and SEQ ID
NO:17.
5. A pair of oligonucleotide primers for amplifying a polymorphic
region in a nucleic acid having a sequence as set forth in SEQ ID
NO:1 from a biological sample, wherein the region corresponds to a
polymorphic site selected from the group consisting of position 245
of SEQ ID NO:1 and position 1470 of SEQ ID NO:1
6. The primers of claim 5, having sequences selected from the group
consisting of: SEQ ID NO:18 and SEQ ID NO:19; and SEQ ID NO:20 and
SEQ ID NO:21.
7. A kit comprising at least one oligonucleotide primer pair
complementary to a polymorphic region of a nucleic acid having a
sequence as set forth in SEQ ID NO:1, wherein the region
corresponds to a polymorphic site selected from the group
consisting of position 245 of SEQ ID NO:1 and position 1470 of SEQ
ID NO:1.
8. The kit of claim 7, comprising at least two oligonucleotide
primer pairs, wherein each primer pair is complementary to a
different polymorphic region of the nucleic acid of SEQ ID
NO:1.
9. A method of diagnosing predisposition to low spine bone mineral
density, low total hip bone mineral density, low femoral neck bone
mineral density, or osteoporosis in a human, said method comprising
the steps of: obtaining a nucleic acid sample from the human; and
detecting the presence or absence of at least one allelic variant
of a polymorphic region in a nucleic acid having a sequence as set
forth in SEQ ID NO:1 in the sample, wherein the polymorphic region
corresponds to the polymorphic site at position 245 of SEQ ID
NO:1.
10. A method of diagnosing predisposition to low total hip bone
mineral density, low femoral neck bone mineral density, or
osteoporosis in a human, said method comprising the steps of:
obtaining a nucleic acid sample from the human; and detecting the
presence or absence of at least one allelic variant of a
polymorphic region in a nucleic acid having a sequence as set forth
in SEQ ID NO:1 in the sample, wherein the polymorphic region
corresponds to the polymorphic site at position 1470 of SEQ D NO:1.
Description
[0001] The present invention relates to oligonucleotides, kits,
microarrays, and methods for detection of bone disease, in
particular, osteopenia and osteoporosis.
BACKGROUND OF THE INVENTION
[0002] Osteoporosis is a disease characterized by low bone mass and
microarchitectural deterioration of bone tissue, leading to
enhanced bone fragility and consequently increased bone fracture.
The cost of osteoporotic fracture in the US alone is estimated at
$13.8 billion per annum. Fracture is the clinical endpoint in
osteoporosis, but bone mineral density (BMD) is commonly used as a
surrogate for determining risk of fracture. BMD is an estimate of
the mineral mass, corrected for the area (anteroposterior
projection) of the bone under study. BMD is the strongest predictor
of osteoporotic fracture known, and this measurement is made using
Dual X-Ray Absorptiometry (DEXA). Data derived from DEXA scans of
the lumbar spine (L1-L4) and hip (total hip and femoral neck) can
be used in studies of osteoporosis.
[0003] In women, peak bone mass is reached between age 20-30, after
which there is a period of consolidation and gradual loss. However,
there is a rapid decline in BMD of approximately 3% per year for
approximately 5 years after menopause. Consequently, estrogen is
strongly implicated as a crucial element in the maintenance of bone
mass. This is supported by the role of hormone replacement therapy
in the prevention and treatment of osteoporosis. A role for
estrogen has also been implicated in the growth and maintenance of
bone in men.
[0004] Twin and family resemblance studies show that osteoporosis
has a substantial genetic component. Data shows that bone mineral
density (BMD) has a heritability in the range of 0.66 to 0.82.
Other data on Colles' (wrist) fracture in families, also supports
the genetic basis of osteoporotic fracture. This data shows that
the relative risk of Colles' fracture, if a woman's mother or
sisters have had a Colles' fracture, is 1.3 and 1.9 respectively.
On the basis of this, and other published data, osteoporosis is
regarded as a complex multifactorial disease with multiple gene and
environmental factors contributing to disease susceptibility.
Lifestyle changes such as moderate exercise; ensuring sufficient
calcium intake, limiting alcohol intake; and cessation of smoking,
are indicated for individuals with osteopenia and osteoporotic bone
disease. Osteopenia and osteoporosis can be treated using lifestyle
changes, hormone replacement therapy (HRT: estrogen), selective
estrogen receptor modulators (SERMs), antiresorptive agents
(bisphosphonates), calcitonin and sodium fluoride. The optimal
treatment for osteoporosis has not been determined.
[0005] A number of genes have been identified which are associated
with predisposition to osteoporosis. For example, U.S. Pat. No.
5,922,542 discloses an association between risk of developing
osteoporosis and certain polymorphisms in the gene encoding
collagen 1.alpha.1, also referred to as COL1A1, and a kit based on
this association has been commercialized in Europe.
[0006] U.S. Pat. No. 5,998,137 discloses methods of diagnosing a
number of diseases, including osteoporosis, by detecting a
polymorphism in the promoter of the transforming growth factor
.beta. (TGF-.beta.) gene. WO 00/23618 discloses a method of
detecting predisposition to osteoporosis on the basis of a
polymorphism in intron 5 of the TGF-.beta. gene. JP2000270897 also
discloses a method for detection of danger of osteoporosis based on
a polymorphism in the TGF-.beta. gene.
[0007] U.S. Pat. No. 5,698,399 discloses methods of diagnosing
predisposition to osteoporosis by detecting certain polymorphic
variants in the gene encoding interleukin-1 receptor
antagonist.
[0008] U.S. Pat. No. 6,066,450 discloses methods for detecting
predisposition to osteoporosis by identifying certain polymorphic
variants in the interleukin-6 gene.
[0009] EP 1054066 discloses a method for determining sensitivity to
an osteoporosis medication based on analysis of certain
polymorphisms in the genes encoding vitamin D receptor, the
estrogen receptor, and apolipoprotein E.
[0010] WO 01/09383 suggests that polymorphisms in the human gene
encoding the melatonin-related receptor, a G-protein coupled
receptor of unknown function, may be involved in bone-related
disorders, including osteoporosis.
[0011] WO 01/23559 suggests that mutations in the regulatory region
of the human gene encoding osteoclast differentiation factor may be
used to detect or predict susceptibility to bone diseases,
including osteoporosis.
[0012] WO 01/20031 discloses that certain single nucleotide
polymorphisms (SNPs) in the human klotho gene are associated with
forearm and spine bone mineral density and thus may be used to
diagnose predisposition to osteoporosis.
[0013] Because osteoporosis is likely to be the result of genetic
variations in multiple genes, additional genes and polymorphisms
which may be associated with this condition are the subject of
ongoing research. A need remains for identification of such genes,
to enhance physicians' ability to detect osteopenia and
predisposition to osteoporosis in individuals who may have the
condition, but do not exhibit symptoms. Thus lifestyle changes or
therapy can be initiated early in the progression of disease.
SUMMARY OF THE INVENTION
[0014] On the basis of genetic analysis of dizygotic twins and sib
pairs, the present inventors have discovered that certain
polymorphisms in the nucleic acid set forth in SEQ ID NO:1 are
associated with variation in bone mineral density (BMD).
Specifically, the presence of an A nucleotide at position 245 of
SEQ ID NO:1 is associated with low spine BMD, low total hip BMD,
and low femoral neck BMD, and the presence of a C nucleotide at
position 245 of SEQ ID NO:1 is associated with higher spine, total
hip, and femoral neck BMD. In addition, the presence of a G
nucleotide at position 1470 of SEQ ID NO:1 is associated with low
total hip and femoral neck BMD, and the presence of an A nucleotide
at position 1470 of SEQ ID NO:1 is associated with higher total hip
and femoral neck BMD. Moreover, the presence of a haplotype
represented by an A nucleotide at position 245 of SEQ ID NO:1 and a
G nucleotide at position 1470 of SEQ ID NO:1 is associated with low
spine BMD, low total hip BMD, and low femoral neck BMD. These
associations may be used as the basis of reagents, kits, and
methods for detection of predisposition to osteoporosis.
[0015] The nucleic acid of SEQ ID NO:1 is a draft human genomic
sequence of nucleotides 58007412 to 58016140 of chromosome 3 (Human
Genome Project Working Draft, University of California, Santa Cruz,
April 2001 Freeze), which comprises a cDNA (GenBank Accession No.
XM.sub.--003213) corresponding to a gene of unknown function
currently known as E2IG3. The complete genomic sequence of E2IG3 is
available in a chromosome 3 working draft sequence with GenBank
Accession No. NT.sub.--005986 [gi:15297785]. E2IG3 is upregulated
by 17.beta. estradiol (estrogen). The protein encoded by E2IG3 is
believed to belong to a subfamily of large GTP-binding proteins.
The E2IG3 cDNA is also disclosed as SEQ BD NO:247 of WO 00/55350
and as SEQ ID NO: 6137 of WO 00/58473.
[0016] In one embodiment, the invention provides a sequence
determination oligonucleotide complementary to a polymorphic region
within a nucleic acid having a sequence as set forth in SEQ ID
NO:1, wherein the region corresponds to a polymorphic site selected
from the group consisting of position 245 of SEQ ID NO:1 and
position 1470 of SEQ ID NO:1.
[0017] In another-embodiment, the invention provides a microarray
comprising at least one oligonucleotide complementary to a
polymorphic region in the nucleic acid set forth in SEQ ID NO:1,
wherein the region corresponds to a polymorphic site selected from
the group consisting of position 245 of SEQ ID NO:1 and position
1470 of SEQ ID NO:1.
[0018] In another embodiment, the invention provides the
oligonucleotide primer pairs useful for amplification of a
polymorphic region in the nucleic acid of SEQ ID NO:1 from a
biological sample, wherein the region corresponds to a polymorphic
site selected from the group consisting of position 245 of SEQ ID
NO:1 and position 1470 of SEQ ID NO:1.
[0019] In another embodiment, the invention provides a kit
comprising at least one oligonucleotide primer pair complementary
to a polymorphic region of the nucleic acid of SEQ ID NO:1, wherein
the region corresponds to a polymorphic site selected from the
group consisting of position 245 of SEQ ID NO:1 and position 1470
of SEQ ID NO:1.
[0020] The invention is also embodied in a method of diagnosing
predisposition to low spine bone mineral density, low total hip
bone mineral density, low femoral neck bone mineral density, or
osteoporosis in a human, said method comprising the steps of
obtaining a nucleic acid sample from the human; detecting the
presence or absence of at least one allelic variant of a
polymorphic region in a nucleic acid having a sequence as set forth
in SEQ ID NO:1 in the sample, wherein the polymorphic region
corresponds to the polymorphic site at position 245 of SEQ ID
NO:1.
[0021] The invention is also embodied in a method of diagnosing
predisposition to low total hip bone mineral density, low femoral
neck bone mineral density, or osteoporosis in a human, said method
comprising the steps of obtaining a nucleic acid sample from the
human; and detecting the presence or absence of at least one
allelic variant of a polymorphic region in a nucleic acid having a
sequence as set forth in SEQ ID NO:1 in the sample, wherein the
polymorphic region corresponds to the polymorphic site at position
1470 of SEQ ID NO:1.
[0022] In a further embodiment, the invention provides a method of
diagnosing predisposition to low spine bone mineral density, low
total hip bone mineral density, low femoral neck neck bone mineral
density, or osteoporosis in a human comprising the steps of
obtaining a nucleic acid sample from the human; and detecting the
presence or absence of a haplotype of the nucleic acid having a
sequence as set forth in SEQ ID NO:1, said haplotype being
characterized by: an A nucleotide at position 245 of SEQ ID NO:1
and a G nucleotide at position 1470 of SEQ ID NO:1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 sets forth the nucleic acid of SEQ ID NO:1 with the
polymorphic sites at positions 245 and 1470 in bold capital type.
The transcription start site of E2IG3 is indicated in italics,
introns are depicted in lower case type, and exons are depicted in
bold lower case type. Exons are as described in GenBank Accession
No. XM.sub.--003213.
[0024] FIG. 2 sets forth the sequences of certain oligonucleotides
of the invention, which are correlated with the polymorphic site
the oligonucleotides are designed to detect. Polymorphic sites in
these oligonucleotides are indicated in bold capital type.
[0025] FIG. 3 sets forth the sequences of certain oligonucleotide
primer pairs designed to amplify polymorphic regions of the nucleic
acid of SEQ ID NO:1.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The U.S. patents and publications referenced herein are
hereby incorporated by reference. Examples 1 and 2 below
demonstrate associations between certain polymorphic regions in SEQ
ID NO:1 and measurements of bone mineral density.
[0027] For the purposes of the invention, certain terms are defined
as follows.
[0028] "Oligonucleotide" means a nucleic acid molecule preferably
comprising from about 8 to about 50 covalently linked nucleotides.
More preferably, an oligonucleotide of the invention comprises from
about 8 to about 35 nucleotides. Most preferably, an
oligonucleotide of the invention comprises from about 10 to about
25 nucleotides. In accordance with the invention, the nucleotides
within an oligonucleotide may be analogs or derivatives of
naturally occurring nucleotides, so long as oligonucleotides
containing such analogs or derivatives retain the ability to
hybridize specifically within the polymorphic region containing the
targeted polymorphism. Analogs and derivatives of naturally
occurring oligonucleotides within the scope of the present
invention are exemplified in U.S. Pat. Nos. 4,469,863; 5,536,821;
5,541,306; 5,637,683; 5,637,684; 5,700,922; 5,717,083; 5,719,262;
5,739,308; 5,773,601; 5,886,165; 5,929,226; 5,977,296; 6,140,482;
WO 00/56746; WO 01/14398, and the like. Methods for synthesizing
oligonucleotides comprising such analogs or derivatives are
disclosed, for example, in the patent publications cited above and
in U.S. Pat. Nos. 5,614,622; 5,739,314; 5,955,599; 5,962,674;
6,117,992; in WO 00/75372, and the like. The term
"oligonucleotides" as defined herein includes compounds which
comprise the specific oligonucleotides disclosed herein, covalently
linked to a second moiety. The second moiety may be an additional
nucleotide sequence, for example, a tail sequence such as a
polyadenosine tail or an adaptor sequence, for example, the phage
M13 universal tail sequence, and the like. Alternatively, the
second moiety may be a non-nucleotidic moiety, for example, a
moiety which facilitates linkage to a solid support or a label to
facilitate detection of the oligonucleotide. Such labels include,
without limitation, a radioactive label, a fluorescent label, a
chemiluminescent label, a paramagnetic label, and the like. The
second moiety may be attached to any position of the specific
oligonucleotide, so long as the oligonucleotide retains its ability
to hybridize to the polymorphic regions described herein.
[0029] A polymorphic region as defined herein is a portion of a
genetic locus that is characterized by at least one polymorphic
site. A genetic locus is a location on a chromosome which is
associated with a gene, a physical feature, or a phenotypic trait.
A polymorphic site is a position within a genetic locus at which at
least two alternative sequences have been observed in a population.
A polymorphic region as defined herein is said to "correspond to" a
polymorphic site, that is, the region may be adjacent to the
polymorphic site on the 5' side of the site or on the 3' side of
the site, or alternatively may contain the polymorphic site. A
polymorphic region includes both the sense and antisense strands of
the nucleic acid comprising the polymorphic site, and may have a
length of from about 100 to about 5000 base pairs. For example; a
polymorphic region may be all or a portion of a regulatory region
such as a promoter, 5' UTR, 3' UTR, an intron, an exon, or the
like. A polymorphic or allelic variant is a genomic DNA, cDNA, mRNA
or polypeptide having a nucleotide or amino acid sequence that
comprises a polymorphism. A polymorphism is a sequence variation
observed at a polymorphic site, including nucleotide substitutions
(single nucleotide polymorphisms or SNPs), insertions, deletions,
and microsatellites. Polymorphisms may or may not result in
detectable differences in gene expression, protein structure, or
protein function. Preferably, a polymorphic region of the present
invention has a length of about 1000 base pairs. More preferably, a
polymorphic region of the invention has a length of about 500 base
pairs. Most preferably, a polymorphic region of the invention has a
length of about 200 base pairs.
[0030] A haplotype as defined herein is a representation of the
combination of polymorphic variants in a defined region within a
genetic locus on one of the chromosomes in a chromosome pair. A
genotype as used herein is a representation of the polymorphic
variants present at a polymorphic site.
[0031] A polymorphic region of the present invention comprises a
portion of SEQ ID NO:1 corresponding to at least one of the
polymorphic sites identified above. That is, a polymorphic region
of the invention may include a nucleotide sequence surrounding
and/or including any of the polymorphic sites at positions 245 and
1470 of SEQ ID NO:1. Polymorphic regions in the antisense nucleic
acid complementary to SEQ ID NO:1 are also encompassed in the
present invention, wherein the region includes a nucleotide
sequence surrounding and/or including any of the antisense
positions corresponding to positions 245 and 1470 of SEQ ID NO:1.
FIG. 2 sets forth exemplary oligonucleotides within the scope of
this embodiment. For example, a polymorphic region corresponding to
the polymorphic site at position 245 of SEQ ID NO:1 may comprise a
sequence as set forth in any of SEQ ID NO:2; SEQ ID NO:3; SEQ ID
NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; or SEQ ID
NO:9. A polymorphic region corresponding to the polymorphic site at
position 1470 of SEQ ID NO:1 may comprise a sequence as set forth
in any of SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:13;
SEQ ID NO:14; SEQ ID NO:15; SEQ ID NO:16; or SEQ ID NO:17.
[0032] In certain embodiments of the invention, oligonucleotides
are used as probes for the polymorphic regions in the nucleic acid
having the sequence set forth in SEQ ID NO:1. These
oligonucleotides may also be termed "sequence determination
oligonucleotides" within the scope of the invention, and may be
used to determine the presence or absence of a particular
nucleotide at a particular polymorphic site within the nucleic acid
of SEQ ID NO:1. Specific oligonucleotides of the invention include
any oligonucleotide complementary to any of the polymorphic regions
described above.
[0033] Those of ordinary skill will recognize that oligonucleotides
complementary to the polymorphic regions described herein must be
capable of hybridizing to the polymorphic regions under conditions
of stringency such as those employed in primer extension-based
sequence determination methods, restriction site analysis, nucleic
acid amplification methods, ligase-based sequencing methods,
methods based on enzymatic detection of mismatches,
microarray-based sequence determination methods, and the like. The
oligonucleotides of the invention may be synthesized using known
methods and machines, such as the ABI.TM.3900 High Throughput DNA
Synthesizer and the EXPEDITE.TM. 8909 Nucleic Acid Synthesizer,
both of which are available from Applied Biosystems (Foster City,
Calif.).
[0034] The oligonucleotides of the invention may be used, without
limitation, as in situ hybridization probes or as components of
diagnostic assays. Numerous oligonucleotide-based diagnostic assays
are known. For example, primer extension-based nucleic acid
sequence detection methods are disclosed in U.S. Pat. Nos.
4,656,127; 4,851,331; 5,679,524; 5,834,189; 5,876,934; 5,908,755;
5,912,118; 5,976,802; 5,981,186; 6,004,744; 6,013,431; 6,017,702;
6,046,005; 6,087,095; 6,210,891; WO 01/20039; and the like. Primer
extension-based nucleic acid sequence detection methods using mass
spectrometry are described in U.S. Pat. Nos. 5,547,835; 5,605,798;
5,691,141; 5,849,542; 5,869,242; 5,928,906; 6,043,031; 6,194,144,
and the like. The oligonucleotides of the invention are also
suitable for use in ligase-based sequence determination methods
such as those disclosed in U.S. Pat. Nos. 5,679,524 and 5,952,174,
WO 01/27326, and the like. The oligonucleotides of the invention
may be used as probes in sequence determination methods based on
mismatches, such as the methods described in U.S. Pat. Nos.
5,851,770; 5,958,692; 6,110,684; 6,183,958; and the like. In
addition, the oligonucleotides of the invention may be used in
hybridization-based diagnostic assays such as those described in
U.S. Pat. Nos. 5,891,625; 6,013,499; and the like.
[0035] The oligonucleotides of the invention may also be used as
components of a diagnostic microarray. Methods of making and using
oligonucleotide microarrays suitable for diagnostic use are
disclosed in U.S. Pat. Nos. 5,492,806; 5,525,464; 5,589,330;
5,695,940; 5,849,483; 6,018,041; 6,045,996; 6,136,541; 6,142,681;
6,156,501; 6,197,506; 6,223,127; 6,225,625; 6,229,911; 6,239,273;
WO 00/52625; WO 01/25485; WO 01/29259; and the like. Preferably,
the microarray of the invention comprises at least one
oligonucleotide complementary to a polymorphic region of SEQ ID
NO:1, wherein the region corresponds to a polymorphic site selected
from the group consisting of position 245 of SEQ ID NO:1 and
position 1470 of SEQ ID NO:1. More preferably, the microarray of
the invention comprises an oligonucleotide complementary to a
polymorphic region corresponding to position 245 of SEQ ID NO:1 and
an oligonucleotide complementary to a polymorphic region
corresponding to position 1470 of SEQ ID NO:1. In a specific
embodiment, the oligonucleotides of the microarray of the invention
are complementary to any or all of the polymorphic regions selected
from the group consisting of SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4;
SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ D NO:8; and SEQ ID NO:9
(corresponding to the polymorphic site at position 245 of SEQ ID
NO:1); and the polymorphic regions selected from the group
consisting of SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:12; SEQ ID
NO:13; SEQ ID NO:14; SEQ ID NO:15; SEQ ID NO:16; and SEQ ID NO:17
(corresponding to the polymorphic site at position 1470 of SEQ ID
NO:1).
[0036] The invention is also embodied in oligonucleotide primer
pairs suitable for use in the polymerase chain reaction (PCR) or in
other nucleic acid amplification methods. Each oligonucleotide
primer pair of the invention is complementary to a polymorphic
region of the nucleic acid of SEQ ID NO:1. Thus an oligonucleotide
primer pair of the invention is complementary to a polymorphic
region characteristic of at least one of the polymorphic sites at
positions 245 and 1470 of SEQ ID NO:1. Those of ordinary skill will
be able to design suitable oligonucleotide primer pairs using
knowledge readily available in the art, in combination with the
teachings herein. Specific oligonucleotide primer pairs of this
embodiment include the oligonucleotide primer pairs set forth in
SEQ ID NO:18 and SEQ ID NO:19, which are suitable for amplifying
the polymorphic region corresponding to the polymorphic site at
position 245 of SEQ ID NO:1; and the oligonucleotide primer pairs
set forth in SEQ ID NO:20 and SEQ ID NO:21, which are suitable for
amplifying the polymorphic region corresponding to the polymorphic
site at position 1470 of SEQ ID NO:1. Those of skill will recognize
that other oligonucleotide primer pairs suitable for amplifying the
polymorphic regions of the nucleic acid of SEQ ID NO:1 can be
designed without undue experimentation. In particular,
oligonucleotide primer pairs suitable for amplification of larger
portions of SEQ ID NO:1 Would be preferred for haplotype
analysis.
[0037] Each of the PCR primer pairs of the invention may be used in
any PCR method. For example, a PCR primer pair of the invention may
be used in the methods disclosed in U.S. Pat. Nos. 4,683,195;
4,683,202, 4,965,188; 5,656,493; 5,998,143; 6,140,054; WO 01/27327;
WO 01/27329; and the like. The PCR pairs of the invention may also
be used in any of the commercially available machines that perform
PCR, such as any of the GENEAMP.RTM. Systems available from Applied
Biosystems.
[0038] The invention is also embodied in a kit comprising at least
one oligonucleotide primer pair of the invention. Preferably, the
kit of the invention comprises at least two oligonucleotide primer
pair, wherein each primer pair is complementary to a different
polymorphic region of the nucleic acid of SEQ ID NO:1. More
preferably, the kit of the invention comprises at least one
oligonucleotide primer pair suitable for amplification of
polymorphic regions corresponding to positions 245 or 1470 of SEQ
ID NO:1. This embodiment may optionally further comprise a sequence
determination oligonucleotide for detecting a polymorphic variant
at any or all of the polymorphic sites corresponding to positions
245 and 1470 of SEQ ID NO:1. The kit of the invention may also
comprise a polymerizing agent, for example, a thermostable nucleic
acid polymerase such as those disclosed in U.S. Pat. Nos.
4,889,818; 6,077,664, and the like. The kit of the invention may
also comprise chain elongating nucleotides, such as dATP, dTTP,
dGTP, dCTP, and dITP, including analogs of dATP, dTTP, dGTP, dCTP
and dITP, so long as such analogs are substrates for a thermostable
nucleic acid polymerase and can be incorporated into a growing
nucleic acid chain. The kit of the invention may also include chain
terminating nucleotides such as ddATP, ddTTP, ddGTP, ddCTP, and the
like. In a preferred embodiment, the kit of the invention comprises
at least one oligonucleotide primer pair, a polymerizing agent,
chain elongating nucleotides, at least one sequence determination
oligonucleotide and at least one chain terminating nucleotide. The
kit of the invention may optionally include buffers, vials,
microtiter plates, and instructions for use.
[0039] Methods of diagnosing predisposition to low spine bone
mineral density, low total hip bone mineral density, low femoral
neck bone mineral density, or osteoporosis in a human are also
encompassed by the present invention. In the methods of the
invention, the presence or absence of at least one polymorphic
variant of the nucleic acid of SEQ ID NO:1 is detected to determine
or diagnose such a predisposition. Specifically, in a first step, a
nucleic acid is isolated from biological sample obtained from the
human. Any nucleic-acid containing biological sample from the human
is an appropriate source of nucleic acid for use in the methods of
the invention. For example, nucleic acid can be isolated from
blood, saliva, sputum, urine, cell scrapings, biopsy tissue, and
the like. In a second step, the nucleic acid is assayed for the
presence or absence of at least one allelic variant of any or all
of the polymorphic regions of the nucleic acid of SEQ ID NO:1
described above. Preferably, the polymorphic regions on both
chromosomes in the chromosome pair of the human are assayed in the
method of the invention, so that the zygosity of the individual for
the particular polymorphic variant may be determined.
[0040] Any method may be used to assay the nucleic acid, that is,
to determine the sequence of the polymorphic region, in this step
of the invention. For example, any of the primer extension-based
methods, ligase-based sequence determination methods,
mismatch-based sequence determination methods, or microarray-based
sequence determination methods described above may be used, in
accordance with the present invention. Alternatively, such methods
as restriction fragment length polymorphism (RFLP) detection,
single strand conformation polymorphism detection (SSCP), PCR-based
assays such as the TAQMAN.RTM. PCR System (Applied Biosystems) may
be used.
[0041] In accordance with one method of the invention,
predisposition to low spine bone mineral density, low total hip
bone mineral density, low femoral neck bone mineral density, or
osteoporosis is diagnosed by determining the identity of the
nucleotide at position 245 of SEQ ID NO:1. In this method, an A
nucleotide at position 245 of SEQ ID NO:1, or a T nucleotide at the
corresponding position of the antisense complement of SEQ ID NO:1,
is indicative of greater risk of developing low spine bone mineral
density, low total hip bone mineral density, low femoral neck bone
mineral density, or osteoporosis.
[0042] Conversely, the presence of a C nucleotide at position 245
of SEQ ID NO:1, or of a G nucleotide at the corresponding position
of the antisense complement of SEQ ID NO:1, is indicative of a
lower risk of developing low spine bone mineral density, low total
hip bone mineral density, low femoral neck bone mineral density, or
osteoporosis. In a further step, the zygosity of the individual may
be determined, wherein a homozygous AA genotype at position 245 of
SEQ ID NO:1 or TT genotype at the corresponding position of the
antisense complement of SEQ ID NO:1, indicates greatest risk for
developing low spine bone mineral density, low total hip bone
mineral density, low femoral neck bone mineral density, or
osteoporosis. A person whose genotype is homozygous CC at position
245 of SEQ ID NO:1 or GG at the corresponding position of the
antisense complement of SEQ ID NO:1 is at least risk for developing
low spine bone mineral density, low total hip bone mineral density,
low femoral neck bone mineral density, or osteoporosis. An
individual whose genotype is heterozygous AC at position 245 of SEQ
ID NO:1 or TG at the corresponding position of the antisense
complement of SEQ ID NO:1 is at intermediate risk for developing
low spine bone mineral density, low total hip bone mineral density,
low femoral neck bone mineral density, or osteoporosis.
Alternatively, predisposition to low total hip bone mineral
density, low femoral neck bone mineral density, or osteoporosis is
diagnosed by determining the identity of the nucleotide at position
1470 of SEQ ID NO:1. In this embodiment, predisposition to low
total hip bone mineral density, low femoral neck bone mineral
density, or osteoporosis is diagnosed by determining the identity
of the nucleotide at position 1470 of SEQ ID NO:1. In this method,
a G nucleotide at position 1470 of SEQ ID NO:1, or a C nucleotide
at the corresponding position of the antisense complement of SEQ ID
NO:1, is indicative of greater risk of developing low total hip
bone mineral density, low femoral neck bone mineral density, or
osteoporosis. Conversely, the presence of an A nucleotide at
position 1470 of SEQ ID NO:1, or of a T nucleotide at the
corresponding position of the antisense complement of SEQ ID NO:1,
is indicative of a lower risk of developing low total hip bone
mineral density, low femoral neck bone mineral density, or
osteoporosis. In a further step, the zygosity of the individual may
be determined, wherein a homozygous GG genotype at position 1470 of
SEQ D NO: 1 or CC genotype at the corresponding position of the
antisense complement of SEQ ID NO:1, indicates greatest risk for
developing low total hip bone mineral density, low femoral neck
bone mineral density, or osteoporosis. A person whose genotype is
homozygous AA at position 1470 of SEQ ID NO:1 or TT at the
corresponding position of the antisense complement of SEQ ID NO:1
is at least risk for developing low total hip bone mineral density,
low femoral neck bone mineral density, or osteoporosis. An
individual whose genotype is heterozygous GA at position 1470 of
SEQ ID NO:1 or CT at the corresponding position of the antisense
complement of SEQ ID NO:1 is at intermediate risk for developing
low total hip bone mineral density, low femoral neck bone mineral
density, or osteoporosis. In another method of the invention, risk
of low spine bone mineral density, low total hip bone mineral
density, low femoral neck bone mineral density, or osteoporosis is
assessed by determining the haplotype of the individual for both
polymorphic positions within SEQ ID NO:1. For example, individuals
who possess the SEQ ID NO:1 haplotype characterized by an A
nucleotide at position 245 of SEQ ID NO:1 and a G nucleotide at
position 1470 of SEQ ID NO:1 are at higher risk of development of
low spine bone mineral density, low total hip bone mineral density,
low femoral neck bone mineral density, or osteoporosis. This
haplotype may alternatively be detected on the antisense complement
of SEQ ID NO:1 as a T nucleotide at the antisense position
corresponding to position 245 of SEQ ID NO:1 and a C nucleotide at
the antisense position corresponding to position 1470 of SEQ ID
NO:1. Individuals who are homozygous for allelic variants
comprising this haplotype are at particularly high risk of
developing low spine bone mineral density, low total hip bone
mineral density, low femoral neck bone mineral density, or
osteoporosis.
[0043] The examples set forth below are provided as illustration
and are not intended to limit the scope and spirit of the invention
as specifically embodied therein.
EXAMPLE 1
Clinical Samples
[0044] A study design based on extreme discordant and concordant
sib pairs (EDACS) was chosen for analysis. All available female
sibs of families containing EDACS were included in the sample.
EDACS were defined on the criteria of BMD Z score. In each family,
probands required a BMD Z for total spine (L1-4), total hip or
femoral neck of Z.ltoreq.-1.5. At least one additional sib in the
family was required to have either BMD for total spine (L1-4),
total hip or femoral neck equal to Z.ltoreq.-1.0 or Z.ltoreq.1.0. Z
scores were derived from individual BMD scan data (Hologic) of the
subjects and transformed using the published Hologic reference
range for spine BMD (Favus, 1999 in Primer on the metabolic bone
diseases and disorders of mineral metabolism, Ed. Favus, M J,
4.sup.th Edition, Lippincott Williams and Wilkins, Philadelphia,
USA. 1999:483-484 or NHANESIII (Looker et al. (1995) J Bone Mineral
Res. 10, 796-802) for hip and femoral neck. Any BMD data collected
using Lunar technology was transformed to the Hologic BMD
equivalent using sBMD (Genant et al. (1994) J Bone Mineral Res.
9:1503-14; Steiger et al. (1995) J Bone Mineral Res. 10:1602;
Hanson et al. (1997) J Bone Mineral Res. 12:1316).
[0045] A BMD Z score <-1.5 or >+1.5 corresponds to the bottom
or top 6.7% of the age matched distribution, respectively. A Z
score of -1.0 or +1.0 corresponds to being in the bottom or top
15.9% of the age matched distribution and includes both
osteoporotic and osteopenic individuals. A total of 1098 samples
were selected and plated for analysis as the study sample set.
[0046] Whole genome scans were performed on 1401 twin pairs using
up to 706 highly polymorphic microsatellite markers (Reed et al.
(1994) Nat Genet 7:390-5). Additional whole genome scans were
performed on 649 subjects from 283 families containing a proband
with low bone mineral density. These later subjects were genome
scanned using the Affymetrix HuSNP GENECHIP.RTM. technology
platform (Wilson et al. (2000) Calcified Tissue International
67:484). Multipoint nonparametric linkage analysis was performed
using MAPMAKER/SIBS (Kruglyak & Lander (1995) Amer. J. Human
Genetics 57:439-454).
[0047] For fine mapping and SNP genotyping a study design based on
extreme exclusion criteria for probands and sibs were used and
verified by questionnaire if possible. These exclusion criteria
were myeloma, osteosarcoma, or malignancy with skeletal
involvement, hyperparathyroidism, unstable thyroid disease, long
term steroid use (>5 mg/day for more than 6 months and presently
on therapy), chronic immobility, rheumatoid arthritis, anorexia
nervosa (>1 yr), history of osteomalacia, amenorrhea for >6
months, premature cessation of regular menstruation or surgical
oophorectomy +/-/HRT (age <35 yrs), very late menarche >20
years of age, 15>BMI>40, and epilepsy with use of
anticonvulsant medication for >1 year. These exclusions were
applied conservatively and only subjects who had substantial
evidence that they should be excluded were removed, because it was
not possible to verify the exact details with the patients or the
medical records.
EXAMPLE 2
Genotyping
[0048] The linkage studies used a proprietary bioinformatics
infrastructure and proprietary software packages to record marker
positions, store data and generate data files (See WO 00/51053).
Output from these systems was then used with the relevant
application software to perform the statistical analysis.
[0049] MAPMAKER/SIBS (Kruglyak & Lander (1995) Amer. J Human
Genetics 57, 439-454) was used to estimate multipoint nonparametric
linkage for the fine mapping studies. The computer program QTDT
(Abecasis et al. (2000) Amer. J Human Genetics 66, 279-292) was
used to test for evidence of association. Three tests were
considered: Transmission/disequilibrium test (TDT), population
stratification and total association tests. Population
stratification can lead to spurious results from total association
testing, so the latter result was considered and reported only when
there was no evidence of population stratification.
[0050] Haplotype analysis was with QPDT (Martin et al. (2000) Amer.
J. Human Genetics 67, 146-54), which utilises the EM (Dempster et
al. (1977) J Royal Statistical Soc. B39, 1-38) algorithm to assign
haplotypes based on likelihood maximization.
[0051] A. Microsatellite Fine Mapping
[0052] Thirty-five microsatellite markers in a broad interval (50.4
cM-111 cM) on chromosome 3 were chosen for analysis in the study
population, with a mean spacing of 2.02 cM between each marker,
across the region. Genotyping reactions were generally carried out
in microtitre plates (384-well, reaction volume 5 .mu.l),
containing 12.5 ng of DNA DNA from study subjects was amplified
using PCR and sequence specific oligonucleotide primers labelled
with 6-FAM.TM., HEX.TM., or NED.TM. fluorescent dyes. PCR products
were analysed by electrophoresis in a polyacrylamide denaturing
gel, with an ABI PRISM.TM. GENESCAN.RTM. 400HD ROX labelled size
standard in each lane on an ABI model 377 analyzer (Applied
Biosystems, Foster City, Calif.). For genotyping, the chosen
markers were divided into two groups (panels) so that the analysis
of all of the markers could be performed in two electrophoresis
runs of each sample. Consequently, there was no overlap of fragment
sizes in any one dye for either of the panels. Genotype analysis
was performed using ABI PRISM.TM. GENESCAN.RTM. software (version
3.0), and genotyped manually using ABI PRISM.TM. Genotyper 2.0.
Results were input into a proprietary database and binned by
marker. The results were quality checked, ensuring consistent
inheritance within families. Families that were found to have
consistent pedigree problems were excluded from the analysis
set.
[0053] The ordering of genetic mapping markers (i.e. microsatellite
markers) was relatively stable in the region analyzed according to
the Unified Data Base for Human Genome Mapping, Weizmann Institute
of Science (UDB) and National Center for Biotechnology Information,
National Institutes of Health (NCBI) assemblies during the duration
of the study. Conversion of genetic to physical positions for
strategic microsatellite markers was performed using UDB and NCBI
as the reference standards. Comparisons of the identity and
positioning of genomic contigs in the region were also made between
UDB and NCBI and provided relatively good agreement. A comparison
of the positioning of all identified and predicted genes within the
region was also made between NCBI (build 22) and Joint Project
between European Bioinformatics Institute and the Sanger Centre
(ENSEMBL). At its broadest the region encompassed genomic contigs
NT.sub.--005980.3 to NT.sub.--005607.3 (build 22) or
NT.sub.--005498.4 to NT.sub.--005589.4 (build 24). Given the
identified mapping inconsistencies between public domain genome
assemblies, several additional contigs were also considered. The
major focus within this broad region was between NT.sub.--006022.3
and NT.sub.--005589.3 (NCBI build 22; 43.9 Mb-57.2 Mb).
[0054] The microsatellite marker analysis showed linkage of spine
BMD Z to chromosome 3p21 with a non parametric Z=4.07 at 68.8 cM
(n=1619 pedigrees). Using the -1 LOD approach, the support interval
was 62.16 cM-75.62 cM. Total hip gave a peak Z score within the
region of 1.17 at 65.1 cM and femoral neck Z=1.02 at 60 cM.
[0055] The additional markers increased the information content for
linkage, from a mean of 0.4382 to 0.5623, (range of 0.3177-0.6967).
In the region of greatest interest, from 50 cM to 100 cM,
information content for linkage has mean 0.6007 and range
0.5357-0.6967.
[0056] No association was found using multi-allelic test of
association for microsatellite markers within the support
interval.
[0057] B. SNP Genotyping
[0058] SNPs analysed in the study were sourced from the public SNP
resource, dbSNP, which contains an estimated 1.4 million
non-redundant SNPs mapped to the NCBI genome browser (Stoneking,
2001, Nature 409: 821-822).
[0059] SNPs were selected at approximately 50 kb intervals across
the region analyzed. The following attributes were taken into
account when selecting SNPs: amount of available 5' and 3' sequence
context, absence of repeat masking in surrounding sequence,
presence of multiple submissions, identity and reputation of the
submitter. Relatively high SNP failure rate has been predicted, so
several rounds of SNP selection were planned and undertaken.
[0060] Where SNP validation and screening were performed, the SNP
assay was run on a sample set composed of 96 samples and replicated
in quadruplicate. Results were examined to verify a working PCR
reaction and appropriate PCR product, and to establish that some
level of polymorphism existed.
[0061] Five SNPs in the E2IG3 gene (also known as Q9UJY0) reported
by dbSNP were examined. Two of the SNPs (corresponding to position
245 of SEQ ID NO:1, and position 1470 of SEQ D NO: 1) were
genotyped in the complete sample set. The SNPs were amplified
(GENEAMP.RTM. PCR system 9700, Applied Biosystems) using 4.6 ng of
genomic DNA in a total volume of 22.3 .mu.l per well. Genotyping
was performed using PSQ.TM. 96 SNP Reagents Kit 5x96 and SNP
detection was subsequently performed using the PSQ 96 platform
(Pyrosequencing AB, Uppsala, Sweden). In order to detect any
genotyping anomalies, Hardy-Weinberg, haplotype analysis and
duplicate control genotypings were performed. No deviation in
genotyping results could be seen in the duplicates. The frequency
of the three different genotypes for each SNP did not differ
significantly from expected values in the Hardy-Weinberg test. Both
markers and were found to show significant association by TDT with
total hip BMD Z (p=0.0009 and p=0.0045 respectively). Data for
femoral neck BMD Z also shows significant association with the two
markers (p=0.0424 and p=0.0272 respectively). In comparison, spine
BMD showed only a weak association to spine BMD with p-values 0.058
and 0.061 respectively, although haplotypes analysis did show
association (p=0.0325 for haplotype AG). Analysis with QPDT gave
similar results with the lowest p-value for association being with
the SNP corresponding to position 245 of SEQ ID NO:1 and total hip
BMD, a p-value of 0.0002. The association with spine BMD was 0.016
and 0.076, respectively. Weight is known to explain a substantial
proportion of the variance in BMD at weight bearing sites, and
particularly at the hip. Consequently, weight adjusted hip BMD was
also studied in relation to the E2IG3 association with hip BMD.
After adjustment for weight, evidence of association with total hip
BMD remains in the analysis (e.g. position 245 of SEQ ID NO:1;
p=0.003).
[0062] As a result of these findings fragments covering exon 2, 12,
13 and 14 of E2IG3 were sequenced using solid phase sequencing
(AUTOLOAD.TM. Solid Phase Sequencing kit, Amersham Pharmacia
Biotech) and gel electrophoresis on ALFEXPRESS.TM. sequencers
(Amersham Pharmacia Biotech) to identify additional SNPs, between
the associated SNPs and in the 3' end of the gene. One additional
SNP has been discovered in Exon 13. This is a G to C polymorphism
at position 7840 in SEQ ID NO:1. Preliminary data suggests that the
frequency of this SNP is below 5%.
[0063] While the invention has been described in terms of the
specific embodiments set forth above, those of skill will recognize
that the essential features of the invention may be varied without
undue experimentation and that such variations are within the scope
of the appended claims.
Sequence CWU 1
1
21 1 8778 DNA Homo sapiens 1 ctgctgctat tgctcctccg gccgcggccg
ctgccgtcgc ttcggcaccc gccgccctca 60 cctcccttac ccctcccggt
gccgccgcaa aaccagtccc gcggccgcca agcgatccct 120 gctccgcgcg
acactgcgtg cccgcgcacg cagagaggcg gtgacgcact ttacggcggc 180
agcgtaagtg cgtgacgctc gtcagtggct tcagttcaca cgtggcgcca gcggaggcag
240 gttgmtgtgt ttgtgcttcc ttctacagcc aatatgaaaa ggcctagtaa
gtggggtcgg 300 gaggcgggcg tggagggacc cacgtctgga agttgctgca
gccaccacga cgctcttcta 360 cggctacggc tttgtctctg ctggtatggg
ggtgggagcc tacgcgtagg ccttggccct 420 atttcctggt agaaccgaga
gttggaagtc cctacggcga tcatgttaac cgcgcgggct 480 cattctgcgg
aacgaagccg ggcagagggt ggggaagact aggctagatt ttcgtaagga 540
agcagcgtct gagccaggtt tgaggcccaa tattttcttt ccgtggccac gtgcagactg
600 gcccaggtga gagctgagaa tcgcctccca gactcagtgt tcctctcctg
ccttatgatt 660 cgtgctgttt gacacgaagt ggttgtcgtt ttgtgtctca
tacgctgttg tgtatgatcc 720 cattctaata ttgtgagggt aagtgcaggg
aattttgact ccattctgga tctactgaat 780 ttaattctct gggatttgaa
agtagcacgt atgtttgcat taggcatttc gcattagact 840 taacgttagg
tttggtagcc aataacacaa gaaaaggata taactccata gtgcgttaac 900
ccagaactaa tcatttgggt taacagattt gtgatgtgtt tctttgtaga gttaaagaaa
960 gcaagtaaac gcatgacctg ccataagcgg tataaaatcc aaaaaaaggt
aagtgtagtg 1020 cttgagagag ctgtaccaaa cacattgcta aactgatttt
gccctgttcc tttgcgggaa 1080 agtctgggtt aatgtgattt ggttttggga
aatggcattg gatagactga ccatgggcac 1140 aagctcttag gcatcaggag
tgcagctgtg agaaagtgca gtgatttggt gataagtctc 1200 taaatttgtt
cagcatgtta atctctgcat agagagcctt ctagttacaa tttcttgctg 1260
ttttatactt acatatgcat tactttgtaa gattccaatt aaagctccat tttcctagga
1320 cattttatag gcataactaa attgcagcca gattggtttc tcacttgaat
tctgcttaag 1380 tataaagata tttttgtaag cagacaaaat ctctttattt
taataggttc gagaacatca 1440 tcgaaaatta agaaaggagg ctaaaaagcr
gggtcacaag aagcctagga aagacccagg 1500 agttccaaac agtgctccct
ttaaggaggc tcttcttagg gaagctgagc taaggaaaca 1560 gagggtaagt
tatgttagcc agaattttca ttgagtggtg tagtgtgtta tgtgtgatat 1620
ttttcagagt aaggtaacaa cactagtcac tggttcacct atttccctta tggctctgac
1680 agcttgaaga actaaaacag cagcagaaac ttgacaggca gaaggaacta
gaaaagaaaa 1740 gaaaacttga aactaatcct gatattaagc catcaaatgt
ggaacctatg gaaaaggtat 1800 gattaggtct ctttatgaat gagagatcag
gggttttgat tttggttttt ttgcttgggc 1860 ctgagtgcag tggcacaatc
acaactcact gcaacctgga ccacccaggc tcaagcagtc 1920 ttaccacctc
agtctccaag tagctgggac tacagaagca caccatcacg cctggctaat 1980
tttttttagc agacacgggc tttcactata ttgccaaggc tggtctcaag tgatccaccc
2040 aactcagcct cccgaagttt ctggtattac aggtgtgggc tgttgtgcct
ggctgagaga 2100 tgagttctga tgcagaaata aaagcacatc cacaggctgc
tgagcttctt gggaggaaga 2160 caactgagtt cagactccat cttacctatt
taacaactgc aagggctgct actcagctgt 2220 ggaaaatgga gttagaggtt
acagttgctc tacttctaat tttgtgttat ttcccccttt 2280 atcctctagg
agtttgggct ttgcaaaact gagaacaaag ccaagtcggg caaacagaat 2340
tcaaagaagc tgtactgcca agaacttaaa aaggtatctt agcctaggtc agtgtctgac
2400 agtagtaatg aggtttaaaa gactcaagtc attttttttt taacctttta
agatgaaggg 2460 tgcatgtgca ggtttgttat atgggtaaac ttgcgtcata
ggggtttgct gtacagatta 2520 tttcatcacc caggtattaa gcctagtacg
acattagtta tctttcctga tcctctccct 2580 cctcccacct tttcaccaac
aaatcatttc gtgactcgac tctagcttat gctgtttaat 2640 gcctttcctg
ctatgtttac ctgacggaaa tagtttcttt ggttctaaat atttgcacaa 2700
aactggttct gcctgtaagc atgatttaca acattaaaaa aaaacgttga catagtgttg
2760 agattgagaa aggtacattg gagtaagcag tgtcaggcta aaggtctcta
aagtactctg 2820 ttgaaaccta agtgaaggag gacaacttgg tgtagttgct
ctccagcact cccatcccca 2880 acatccattt tcccaagctc actcccccat
ggatagaacc tgactgcccc tcagcagctt 2940 ttggcaaggc cagaaggacc
tatcaaacta tataattcac tatgggagga ttcagacagg 3000 gatatttgca
tttttgaaat ccatcttgat cagagactgc tgagcaagcc tatgttttac 3060
tttcctgtgt gagaaatgat gagggtcaac attcttcata ccaaagtgaa gacatgagat
3120 ccaactctga gctcaccctg ttgctaaatg gataatgcca gtactctctt
gtggaaggta 3180 ttaccagaac aagggatgta gttctgatca ttttctcctt
gataatgtag ttctggtcat 3240 tttttccttg ataggtgatt gaagcctccg
atgttgtcct agaggtgttg gatgccagag 3300 atcctcttgg ttgcagatgt
cctcaggtag aagaggccat tgtccagagt ggacagaaaa 3360 agctggtact
tatattaaat aaatcaggtg agtaaagagg gtaccctttg tcttctgtgt 3420
acatgggtga ggtacgagga aacagtctga tagtcactga agactgatta gatccaactc
3480 tgatctcagc aaagccagag tacgtgcact ttgccagaga cagtgctagg
cagtggggag 3540 ccaggtgact tttacaactg actcaactgg tttctactat
tcttttgcca ttcagtattt 3600 accatctttt aaataaagag tgtaagctgc
tatacccagc ttattgtgta gtatatttca 3660 tctaggaagt gatgacagtg
tgacaaattc cccacaccta cacaatgtcg ggtattagtt 3720 caagagtgaa
ataaattgga acgtatgtga caaaatattt aaatgaaatg cataattatg 3780
catctgagtt tgagcagcag gaaaaagaaa acccagaaca gagaattaca aagcagaaaa
3840 tgggaatgag atctaaaatt gttgttgggg ttaagaaaca attggctgct
tgggaggctg 3900 aagtgggcac atcacttgag gccaggagtt cgagaaaagc
ctggccaaca cggcgaaacc 3960 ccatctctac taaaatacaa atattagccg
ggcatgatga tgggcacttg tagtcccagc 4020 tactcggaag gctgaggcag
gagaattgct tgaacccggg aggcggaggt tgcagtgagc 4080 cgatactgtg
ccactgcact ccagcctggg caacaacact tcgtctcaaa aatgaagaaa 4140
caattggctg ctagctaaag gtaaattctg gaaacatagc tctagggtta gtagggttgt
4200 aatccagacg ttgagtctgt cctgaagttt tcaagtgagc aatacaaggg
gaattgaaat 4260 agagaagtgc agatgctgag ctctgttaag atacgggcag
tatggtaggg gagcttaccc 4320 tgccctgatt ttctagttaa atccttttga
aaggactggg aaaatgtaaa ccagagtaaa 4380 atctatagtt gccagatttt
gcaaatgcat ctcaacaaaa tagccacatt ggagcaaatg 4440 tctttttctt
tttttctttt tgagatggag gcttgctgtg tcacccaggc tggagtgcag 4500
tggcgccatc tcagctcact gcaagctcca cctcccgggt tcacgccgtt ctcctgcctc
4560 agcctcccga gtagctggga ctacaggtgc ccaccaccac gcccagctaa
tttttttgta 4620 tttttagtag agacggggtt ttatcgtgtt agccaggatg
gtcttgatct cctgaactcg 4680 tgatccgcct gtctcggcct cccaaagtgc
tgggattaca ggcgtgagcc accgtgcctg 4740 gccgcaaatg tcttatttct
aattgctatc agatctggta ccaaaggaga atttggagag 4800 ctggctaaat
tatttgaaga aagaattgcc aacagtggtg ttcagagcct caacaaaacc 4860
aaaggataaa gggaagataa ccaaggtatc ctttattagt ggtaagaaat gtgattcttt
4920 cagattttgg ttgaaatatg atgagtgtac aaaatcttga tttaagtgaa
tgaaaaatta 4980 caagatccaa ctctgatttc agccagagat catctgaaag
gcaatgtagt tatcttaaga 5040 gctgggctct ggagcctgat tgcttggggt
ttgttgaaat ttatcaggta agttgccaga 5100 ataattcaac tatttggaat
tttagcgtgt gaaggcaaag aagaatgctg ctccattcag 5160 aagtgaagtc
tgctttggga aagagggcct ttggaaactt cttggaggtt ttcaggaaac 5220
ttgcagcaaa gccattcggg ttggagtaat tggtgagttt cagttcatta ctttttactt
5280 tttaagtgtt gaaatagtta agaagtttag ccagctctcc aagtgcccaa
gcagcagtgt 5340 atggagttgt tgtcaagtaa aaggctcact caaatactag
cttcttgctt ataccttact 5400 gaaagcacat aaccacacac aatttaaaga
aaaaaactta cacaactgct gccagattca 5460 ggttttttgt tgggactgat
tactgtagga agctggtttc taaaagttct tggtttgttt 5520 gaatttatag
tattttcctg cctttgcatt acttgtgcaa gaaatgaaga aactaaaatt 5580
ggtcttagta ttgaagtgaa gacactgaga tccaactctg atcttgccct aaacatcagg
5640 gaaatggaaa attaggcagt gaaaatttca taagtgccaa catataatgc
tttttatatc 5700 tggatttccc atttatttgt aggtttccca aatgtgggga
aaagcagcat tatcaatagc 5760 ttaaaacaag aacagatgtg taatgttggt
gtatccatgg ggcttacaag gtaaatggag 5820 gtgtccataa ttgtaatatt
atagtgacac actattttat tttggttatc tcaaggaagg 5880 tgattttttt
tttttttttt tttgagacag tttcactctt gttcccaggc tggagggcaa 5940
tggcgcaatc tcggctcact gcaacctcca cctctcaggt tcaagtgatt ctcctgcctc
6000 agcctccaga gtagctggga ttacaggcat gtgcccccac gcccggctaa
ttttgtattt 6060 ttaatagaga cgggtttctc catgttggtt aggctggtct
caaactcctg acttcagctg 6120 atctgcccgc ctcggcctcc caaagtgctg
gggttacagg ccaagccacc gcgcccggcc 6180 tatttttatt ttttaaagct
tttggtgaaa gcagagattt aagccagtgc tagaactgat 6240 tggagtggga
cagctgccca caatttagtt tgaaagacaa gttcagcaga tagattgaga 6300
gagaggaaag ctccctcaga ggaagtgatg tttgaacctg gccttgagaa attaatagaa
6360 atttgccaga tagaagtctg ccaccacacc tggctagttt ttgtattttt
aatagagatg 6420 gggtttcact gttggccagg ctagcctcaa actcctggcc
ttaagtcatc cacccgccta 6480 ggcctcacaa agtgttggga ttacacttgt
gttgggtggc atgagccact gtgcctggcc 6540 aacttttaga tttttttttt
ttgggagatg gagtctgtct cccaggctgg agtacagtgg 6600 tgctatcttg
gctcactgca acctcagcct cctgagtagc tgggatcaaa ggcacccggc 6660
taatttttgt atttttagta gagacagggc tttcatcatg ttggccaggc tggtctcaaa
6720 ctcctgacct caggtgatcc acccacctcg gcctcccaaa gtgctgggat
tacaggcgtg 6780 agccaccgcg cccagccaga aggtggcata tttatagcaa
aggaaatagc atgtgttttg 6840 gttgatgtaa atatataagc tataacatgt
agtgttctct ttagaacagt cgggtatgct 6900 gttacagttt tagataaatg
tgaagcaaat gatgataaac tggatctgac tgactgtgct 6960 gagtctgttc
aatccaaccc tgagcttcat gttctgtctc ttaacctcca aatagaccaa 7020
tgcccccatc aattccatcg ctcttctttc aggagcatgc aagttgtccc cttggacaaa
7080 cagatcacaa tcatagatag tccgagcttc atcgtatctc cacttaattc
ctcctctgcg 7140 cttgctctgc gaagtccagc aagtattgaa gtagtaaaac
cgatggaggc tgccagtgcc 7200 atcctttccc aggctgatgc tcgacaggta
aaaggacccc ttctcatgag ctccttggag 7260 ccatcttctt tcatcataag
cattttgagt agaaaaatct tggaagtgtt ttaaagtact 7320 ggcatgtcag
atgaaggaca gctcctttgt ttggtttttt ttttaaggta gtactgaaat 7380
atactgtccc aggctacagg aattctctgg aattttttac tgtgcttgct cagagaagag
7440 gtatgcacca aaaaggtgga atcccaaatg ttgaaggtgc tgccaaactg
ctgtggtctg 7500 agtggacagg gtaagctttc ttttctgttg gcattttggt
gaccactaga ataaaccttc 7560 ttttgacaca tcttattttt aatatcagtg
cctcattagc ttactattgc catcccccta 7620 catcttggac tcctcctcca
tattttaatg agagtattgt ggtagacatg aaaagcggct 7680 tcaatctgga
agaactggaa aagaacaatg cacagagcat aagaggtgag aattgtgtgt 7740
cgctgctgtc ttcatcagct gacaggccag tggagctctt acctgtttac atgggcttgc
7800 tttctttccc agccatcaag ggccctcatt tggccaatas catccttttc
cagtcttccg 7860 gtctgacaaa tggaataata gaagaaaagg acatacatga
agaattgcca aaacggaaag 7920 aaaggaagca ggaggagagg gaggatgaca
aagacagtga ccaggaaact gttgatgaag 7980 aagttgatgt aagtgtgtcc
tccatgagtt aaaactgaag tgagttttct agcattataa 8040 tacataatgg
aaggaactga agataggaaa tatttgaggc ttgtgatcca ttagccttaa 8100
ttttgcacat cccgttatat gtacctccaa agagttaatt tttcaggtac ataactactt
8160 ggattaaatg agcagacaag ggctactaat ccagcactat ttttctttgt
cacacaggaa 8220 aacagctcag gcatgtttgc tgcagaagag acaggggagg
cactgtctga ggagactaca 8280 gcaggtgagg caggcaaaag gggttctaac
gaagcagcat ggtatagaat cacttttact 8340 ttttgaaaat ctctttattt
tcctgcaata taggtgaaca gtctacaagg tcttttatct 8400 tggataaaat
cattgaagag gatgatgctt atgacttcag tacagattat gtgtaacaga 8460
acaatggctt tttatgattt tttttttaac attttaagca gactgctaaa ctgttctctg
8520 tataagttat ggtatgcatg agctgtgtaa attttgtgaa tatgtattat
attaaaacca 8580 ggcaacttgg aatccctaaa ttctgtaaaa agacaattca
tctcattgtg agtggaagta 8640 gttatctgga ataaaaaaag aagataccta
ttgaaaaatg taagttttat ttacagatca 8700 ggccacaggt tacaaaatta
aaaccaacag cagttttgaa ttatctgtac cagctagctg 8760 aactagccat
atcagttc 8778 2 15 DNA Homo sapiens 2 agcggaggca ggttg 15 3 15 DNA
Homo sapiens 3 ggaagcacaa acaca 15 4 13 DNA Homo sapiens 4
aggttgatgt gtt 13 5 13 DNA Homo sapiens 5 aggttgctgt gtt 13 6 13
DNA Homo sapiens 6 aacacatcaa cct 13 7 13 DNA Homo sapiens 7
aacacagcaa cct 13 8 15 DNA Homo sapiens 8 tgtgtttgtg cttcc 15 9 15
DNA Homo sapiens 9 caacctgcct ccgct 15 10 15 DNA Homo sapiens 10
aggaggctaa aaagc 15 11 15 DNA Homo sapiens 11 ggcttcttgt gaccc 15
12 13 DNA Homo sapiens 12 aaaagcaggg tca 13 13 13 DNA Homo sapiens
13 aaaagcgggg tca 13 14 13 DNA Homo sapiens 14 tgaccctgct ttt 13 15
13 DNA Homo sapiens 15 tgaccccgct ttt 13 16 15 DNA Homo sapiens 16
gggtcacaag aagcc 15 17 15 DNA Homo sapiens 17 gctttttagc ctcct 15
18 20 DNA Homo sapiens 18 ctcgtcagtg gcttcagttc 20 19 22 DNA Homo
sapiens 19 ccttttcata ttggctgtag aa 22 20 22 DNA Homo sapiens 20
aataggttcg agaacatcat cg 22 21 20 DNA Homo sapiens 21 ttggaactcc
tgggtctttc 20
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